Method: Peroxidase activities have traditionally been expressed in units based upon the rate of oxidation of pyrogallol, a method introduced by Willstalter and Stoll in 1917, and which more recent studies have shown to be somewhat inadequate. (Maehly and Chance 1954.) A wide variety of hydrogen donors have been utilized in peroxidase assay systems including potentially carcinogenic compounds such as o-dianisidine. An improved assay has been adopted using 4-aminoantipyrine as hydrogen donor (Trinder 1966). The reaction rate is determined by measuring an increase in absorbance at 510 nm resulting from the decomposition of hydrogen peroxide. One unit results in the decomposition of one micromole of hydrogen peroxide per minute at 25°C and pH 7.0 under the specified conditions.
Dissolve at one mg/ml in reagent grade water. Immediately prior to use, dilute further to obtain a rate of 0.02-0.04 ΔA/min.
Adjust spectrophotometer to 510 nm and 25°C.
Pipette into each cuvette as follows:
Incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml of diluted enzyme and record the increase in A510 for 4-5 minutes. Calculate ΔA510/minute from linear portion of the curve.
Note: Although the reaction rate obtained with the phenolantipyrine method is 4.5-4.7 times less than previous methods, the peroxidase preparations are the same.
The RZ (Reinheitzahl) which is the absorbance ratio, A403/A275, (RZ) has been used as an indication of purity. However, Shannon et al. (1966) report that this ratio for the isozymes varies from 2.50 to 4.19. This, together with the influence exerted by buffer and pH, would seem to render questionable the preciseness of this ratio as a criterion of purity.